摘要
目的构建金黄色葡萄球菌纤连蛋白结合蛋白A(fibronectin binding protein A,FnBPA)r10-11截短体融合蛋白的原核表达菌株并研究其免疫原性。方法采用重组聚合酶链反应(ploymerase chain reaction,PCR)技术从金黄色葡萄球菌Newman株全基因组序列中进行PCR扩增,对扩增产物进行纯化,连接T载体后转入大肠杆菌DH5α中进行克隆,提取重组后的质粒进行双酶切鉴定,并回收目的片段连接到pET-32a质粒中,转化到大肠杆菌BL21(DE3)中进行原核表达,采用HIS蛋白纯化柱纯化截短蛋白FnBPAr10-11,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)鉴定,纯化后的FnBPAr10-11用于免疫小鼠[分为无菌磷酸盐缓冲液(phosphate buffered saline,PBS)组、FnBPA组、FnBPAr10-11组],检测小鼠血清免疫球蛋白G(immunoglobulin G,IgG)水平、细胞因子水平、免疫保护率。结果 SDS-PAGE分析显示,表达后的蛋白相对分子质量约为33.1×103。FnBPAr10-11组与FnBPA组IgG抗体效价达1∶128 000,与PBS组比较差异有统计学意义(P<0.05)。FnBPAr10-11组的细胞因子水平与FnBPA组比较差异无统计学意义(P>0.05),与PBS组比较差异有统计学意义(P<0.01)。攻毒试验中FnBPAr10-11组的免疫保护作用在50%以上。结论成功构建了金黄色葡萄球菌Fn BPAr10-11截短体融合蛋白的原核表达菌株,该截短蛋白免疫原性良好。
Objective To construct a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A(FnBPA) r10-11 truncated fusion protein, and explore the immunogenicity of FnBPAr10-11. Methods Ploymerase chain reaction(PCR) amplification was carried out from the whole genome sequence of Staphylococcus aureus Newman strain by recombinant PCR technique. The amplified product was purified and transformed into Escherichia coli DH5α for cloning. The recombinant plasmid was extracted and identified by double enzyme digestion.The recovered fragment was ligated into the pET-32 a plasmid and transformed into Escherichia coli BL21(DE3) for prokaryotic expression. The FnBPAr10-11 was purified by HIS protein purification column, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), and used to immunize mice, and the mice were divided into phosphate buffered saline(PBS) group, FnBPA group, and FnBPAr10-11 group. The serum levels of immunoglobulin G(IgG) and cytokines, and the immune protection rate of the mice were detected. Results SDS-PAGE result showed that the relative molecular mass of the protein was about 33.1×103. The titers of IgG antibody in FnBPAr10-11 group and FnBPA group reached 1∶128 000, and were significantly different compared with PBS group(P<0.05). The cytokine level in FnBPAr10-11 group was not significantly different compared with that in FnBPA group, and they were extremely significant(P<0.01) compared with that in PBS group. The immuno-protective effect of the FnBPAr10-11 group was over50%. Conclusions The prokaryotic expression strain of Staphylococcus aureu FnBPAr10-11 truncated fusion protein was successfully constructed. The truncated protein has good immunogenicity.
作者
杨汐静
陈晓婷
刘道龙
杨阳
杨光
YANG Xijing;CHEN Xiaoting;LIU Daolong;YANG Yang;YANG Guang(Animal Experiment Center,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China;College of Life Sciences,Heilongjiang Bayi Agricultural University,Daqing,Heilongfiang 163319,P.R.China)
出处
《华西医学》
CAS
2018年第12期1519-1525,共7页
West China Medical Journal
基金
国家自然科学基金(31072120)
